Plant Cell Rep (2014) 33:245–254 DOI 10.1007/s00299-013-1525-z

ORIGINAL PAPER

High contents of very long-chain polyunsaturated fatty acids in different species

Anna K. Beike • Carsten Jaeger • Felix Zink • Eva L. Decker • Ralf Reski

Received: 23 August 2013 / Revised: 19 September 2013 / Accepted: 8 October 2013 / Published online: 30 October 2013 Ó The Author(s) 2013. This article is published with open access at Springerlink.com

Abstract organism Physcomitrella patens, tissue-specific differences Key message have high contents of polyunsat- in the fatty acid compositions between the filamentous urated fatty acids. Tissue-specific differences in fatty protonema and the leafy gametophores were observed. acid contents and fatty acid desaturase (FADS)-encod- These metabolic differences correspond with differential ing gene expression exist. The arachidonic acid-syn- gene expression of fatty acid desaturase (FADS)-encoding thesizing FADS operate in the ER. genes in both developmental stages, as determined via Abstract Polyunsaturated fatty acids (PUFAs) are microarray analyses. Depending on the developmental important cellular compounds with manifold biological stage and the species, AA amounts for 6–31 %, respec- functions. Many PUFAs are essential for the human diet tively, of the total fatty acids. Subcellular localization of and beneficial for human health. In this study, we report on the corresponding FADS revealed the endoplasmic reticu- the high amounts of very long-chain (vl) PUFAs (CC20) lum as the cellular compartment for AA synthesis. Our such as arachidonic acid (AA) in seven moss species. results show that vlPUFAs are highly abundant metabolites These species were established in axenic in vitro culture, as in mosses. Standardized cultivation techniques using pho- a prerequisite for comparative metabolic studies under tobioreactors along with the availability of the P. patens highly standardized laboratory conditions. In the model genome sequence and the high rate of homologous recombination are the basis for targeted metabolic engi- neering in moss. The potential of producing vlPUFAs of Communicated by P. Kumar. interest from mosses will be highlighted as a promising Electronic supplementary material The online version of this area in biotechnology. article (doi:10.1007/s00299-013-1525-z) contains supplementary material, which is available to authorized users. Keywords Physcomitrella patens Polyunsaturated A. K. Beike F. Zink E. L. Decker R. Reski (&) fatty acids Arachidonic acid In vitro cultivation Plant Biotechnology, Faculty of Biology, University of Freiburg, Mosses Metabolite profiling Scha¨nzlestraße 1, 79104 Freiburg, Germany e-mail: [email protected]

C. Jaeger Introduction Core Facility Metabolomics, ZBSA, Center for Biological Systems Analysis, University of Freiburg, Habsburgerstraße 49, Polyunsaturated fatty acids (PUFAs) are ubiquitous 79104 Freiburg, Germany metabolites with a large variety of biological functions. R. Reski Their functions range from key roles in cellular signaling BIOSS-Centre for Biological Signalling Studies, as precursors of hormones and phytohormones to the 79104 Freiburg, Germany maintenance of membrane integrity and dynamics as major components of the biomembrane system. Many very long- R. Reski FRIAS-Freiburg Institute for Advanced Studies, 79104 Freiburg, chain (vl) PUFAs (CC20), especially x-3 PUFAs, are Germany beneficial for human health as they contribute to the 123 246 Plant Cell Rep (2014) 33:245–254 prevention of cardiovascular and inflammatory diseases ability to integrate homologous nucleotide sequences into (Calder 2004; Xue et al. 2013). Vl x-6 PUFAs such as the genome, metabolic engineering, but also the production dihomo-c-linolenic acid (DGLA, 20:3D8,11,14) and arachi- of recombinant proteins, has already been realized in P. donic acid (AA, 20:4D5,8,11,14) as well as the x-3 vlPUFA patens (Bu¨ttner-Mainik et al. 2011; Chodok et al. 2012; eicosapentaenoic acid (EPA, 20:5D5,8,11,14,17) are the pre- Parsons et al. 2012). The high rate of homologous cursors of biologically active signaling compounds in recombination in P. patens is unique among land at humans, namely, eicosanoid hormones, which comprise the current state of knowledge, being comparable with the prostaglandins, leukotrienes and thromboxanes (Samuels- gene targeting efficiency in yeast and several times higher son 1983; Harizi et al. 2008). Eicosanoid hormones than in vascular plants (Strepp et al. 1998; Schaefer 2001; mediate important physiological processes such as hyper- Hohe et al. 2004; Kamisugi et al. 2006). Beside P. patens, sensitivity reactions and inflammatory responses, but also homologous recombination-based gene targeting is also immunity (Samuelsson 1983; Samuelsson et al. 1987; applicable in the moss Ceratodon purpureus (Bru¨cker et al. Harizi et al. 2008). Furthermore, the semi-essential fatty 2005) and the liverwort Marchantia polymorpha (Ishizaki acid AA plays an important role in infant nutrition, as AA et al. 2013), indicating that this might be a common feature levels correlate with first year growth of preterm infants among certain and liverworts, thus expanding (Carlson et al. 1993). the selection of species to be analyzed with regard to Essential PUFAs for the human diet are linoleic acid genetic engineering and the production of metabolites of (LA, 18:2D9,12), a-(ALA, 18:3D9,12,15) and c-linolenic acid interest. (GLA, 18:3 D6,9,12) that need to be ingested via plant-based To quantify the abundance of vlPUFAs among Bry- nutrition, while nutritional sources for AA and EPA are opsida, comparative fatty acid profiles of seven moss spe- mainly marine fishes (Gill and Valivety 1997). However, cies from different phylogenetic groups were established. alternative sources for AA can also be bacteria, fungi The cellular compartment of AA synthesis is the endo- (Yuan et al. 2002), algae (Bigogno et al. 2002) and mosses plasmic reticulum (ER) as confirmed via green fluorescent (Hartmann et al. 1986; Girke et al. 1998; Kaewsuwan et al. protein (GFP)-tagging of the AA-producing FADS from P. 2006). In contrast to mosses which contain large amounts patens. It has previously been shown that the different of vlPUFAs (Grimsley et al. 1981; Hartmann et al. 1986; developmental stages of P. patens protonema and ga- Girke et al. 1998; Zank et al. 2002; Mikami and Hartmann metophores show distinct metabolic profiles for sugar 2004; Kaewsuwan et al. 2006), higher plants rarely possess derivates, amino acids and nitrogen-rich storage com- these as they lack the corresponding enzymes for vlPUFA- pounds (Erxleben et al. 2012). Here, we established com- synthesis (Gill and Valivety 1997). In the moss model parative fatty acid profiles of protonema and gametophores organism, Physcomitrella patens, the genes that encode the to characterize tissue-specific fatty acid contents. The key enzymes of AA synthesis, namely D6- and a D5-fatty observed differences in the PUFA profiles of protonema acid desaturases (FADS) and a D5-fatty acid elongase have and gametophores were compared with and supported by already been identified via targeted gene replacement and microarray-derived gene expression profiles of putative biochemical characterization (Girke et al. 1998; Zank et al. FADS-encoding genes, which for some FADS-coding 2002; Kaewsuwan et al. 2006). Recently, also two P. genes revealed significantly higher expression levels in patens D12-FADS, that are associated with linoleic acid protonema than in gametophores. biosynthesis, were identified and characterized by heter- ologous expression in the yeast Saccharomyces cerevisiae (Chodok et al. 2013). Materials and methods The high abundance of vlPUFAs, which are uncommon in higher plants, marks clear metabolic differences between Plant material and growth conditions mosses and higher plants. On the one hand the use of moss genes in a transgenic approach, e.g., for the optimization of With the exception of the established laboratory strain of P. oil seed crops as an alternative to the use of genes from patens, the moss species were collected in the field and microalgae or fish (Jiao and Zhang 2013), forms a prom- established in axenic in vitro culture as described in Beike ising research field. On the other hand, mosses themselves et al. (2010). The mosses were axenically cultivated on provide the potential for the discovery of yet uncharacter- modified Knop medium (Reski and Abel 1985) under ized metabolites (Cove et al. 2006; Asakawa 2007; Xie and standardized growth conditions of 55–70 lmol m-2 s-1 Lou 2009; Erxleben et al. 2012), but also for the production light intensity and a photoperiod of 16 h light to 8 h dark at of metabolites in the moss bioreactor that was established 23 ± 1 °C (Hohe et al. 2002). Gametophores were grown for cultivation of P. patens (Decker and Reski 2008, 2012). in Petri dishes that were enclosed with NescofilmTM (Roth, Due to the high rate of homologous recombination, i.e., the Karlsruhe, Germany). For vegetative propagation, the 123 Plant Cell Rep (2014) 33:245–254 247

Fig. 1 Moss species selection. Overview of the moss species grown in axenic in vitro culture and analyzed regarding their fatty acid contents. a Physcomitrella patens, b Encalypta streptocarpa, c Pottia lanceolata, d Plagiomnium undulatum, e Atrichum undulatum, f Brachythecium rutabulum, g murale. Scale bar 1mm

gametophores were disrupted with forceps and transferred supernatant was transferred to a new tube with a Pasteur to fresh solid medium. The species collection comprises P. pipette. The remaining pellet was re-extracted with fresh patens, Encalypta streptocarpa, Pottia lanceolata, Pla- chloroform–methanol (2:1 v/v; Folch et al. 1957) con- giomnium undulatum, Brachythecium rutabulum, Rhynch- taining 0.01 % BHT for 10 min at room temperature. After ostegium murale and Atrichum undulatum (Fig. 1). For centrifugation, the supernatants were combined, evaporated fatty acid and RNA extraction the plant material was har- under a stream of nitrogen and dissolved in 1.5 mL chlo- vested with forceps and transferred to liquid nitrogen until roform–methanol (2:1 v/v). Following addition of 0.75 further processing. For fatty acid and RNA extraction from volumes 1 M KCl to remove polar contaminants (Folch protonema, P. patens was grown in liquid Knop medium et al. 1957), the organic phase was isolated and evaporated (Frank et al. 2005), harvested by filtering with a Bu¨chner under a stream of nitrogen. funnel and a vacuum pump, and immediately transferred to Fatty acids were converted into their methyl esters by liquid nitrogen. acidic esterification (Christie 1989). In brief, 1 mL 2.5 % sulfuric acid in methanol was added to the dried organic Fatty acid extraction and GC–MS analysis phase and esterification was carried out for 90 min at 80 °C on a thermomixer. After 5 min at room temperature, Lipid extraction from moss tissue was adapted from Welti 1.5 mL 0.9 % NaCl and 1 mL hexane were added to the et al. (2002). In brief, 100 mg pulverized moss tissue was reaction, from which the organic phase was isolated after transferred into 1 mL 75 °C hot isopropanol containing short mixing and centrifugation. After evaporation under 0.01 % (w/v) butylated hydroxytoluene (BHT) as an anti- nitrogen, fatty acid methyl esters were dissolved in 100 lL oxidant. After shaking the mixture for 15 min at 75 °Cona chloroform and transferred to GC vials. All extraction and thermomixer (Eppendorf, Hamburg, Germany), tubes were derivatization steps were carried out in screw-cap glass centrifuged for 5 min (1,0009g, room temperature) and the tubes sealed with Teflon-coated caps. 1 lL sample aliquots 123 248 Plant Cell Rep (2014) 33:245–254 were injected into an Agilent 7890A/5975C GC–MS sys- according to the manufacturer’s protocol. Complementary tem (Agilent, Waldbronn, Germany). A split/splitless DNA (cDNA) was generated with SuperScript III (Invit- injector was used in pulsed splitless mode at 230 °C and rogen, Karlsruhe, Germany) and PolyT-primers according 9.3 psi pressure. Chromatographic separation was achieved to the manufacturer’s protocol. The CDS were amplified on a 30 m 9 0.25 mm 9 0.25 lm HP-5MS capillary col- from cDNA using oligonucleotides that contained restric- umn (Agilent Technologies, Waldbronn, Germany) with tion enzyme binding sites (165175-GFP-SalI-for: GGTC helium as carrier gas at a flow rate of 1 mL/min. The GACATGGCGCCCCACTCTGCGGAT, 165175-GFP- temperature ramp was programmed as follows: 80 °C for Acc65I-rev: CGGTACCGCCATCGAGCCGAAACTCTG 2 min, 5 °C/min increase to 325 °C, 325 °C held for TC, 164045-GFP-Acc65I-for: GGGTACCGAAATGGTA 10 min. The transfer line connecting GC oven with quad- TTCGCGGGCGGTG, 164045-GFP-BglII-rev: CAGATC rupole MS detector was heated to 260 °C. 70 eV electron TACTGGTGGTAGCATGCTGCTC, 183309-GFP-XhoI-f: impact (EI) mass spectra of eluting compounds were GCTCGAGATGGCGGCCTCTCTGTTGTCCA, 183309- acquired in full-scan mode (m/z 50–500) over a total run- GFP-BglII-r: CAGATCTGAAGGTAGGATCTGTCTGGT time of 61 min. AG). Protoplasts were isolated and transfected as described Peak identification was performed with the AMDIS by Hohe et al. (2004). After transfection, the protoplasts software (Stein 1999) that integrates raw data processing were resuspended in a regeneration medium (Rother et al. (deconvolution, compound detection) and comparison of 1994) and incubated in the dark for 3–4 days before acquired mass spectra/retention times with reference microscopic analysis. libraries. To identify fatty acids, a custom reference library As a control for mitochondria-specific fluorescence pat- was created from a 37-component fatty acid methyl ester terns, the protoplasts were stained with MitoTrackerÒ Orange (FAME) mix (Sigma, Deisenhofen, Germany). In addition, CMTMRos (MTO, Invitrogen, Karlsruhe, Germany), a current versions of the commercial libraries FiehnLib mitochondria-specific fluorescence dye. Before microscopic (Kind et al. 2009) and NIST (NIST 2008) were used. Fatty analysis, 1 lL MTO was added to 1 mL protoplast solution. acids were considered identified when mass spectral simi- After incubation for 10 min, the protoplasts were centrifuged larity between sample and standard was 95 % or higher and at 459g for 10 min. The supernatant was removed, leaving retention times did not deviate more than 3 s. In cases 100 lL for confocal laser scanning electron microscopy. As a where retention time deviation was higher, only chain control for plastid-localization, a putative x-3-FADS pre- length and degree of unsaturation (but not the exact dicted to be localized with 99 % probability and a confidence structural isomer) were determined from the FAME mass of 0.85 in the chloroplasts using YLoc (LowRes Plants) spectrum where possible (Christie 1989). Such fatty acids (Briesemeister et al. 2010) was tagged with GFP. were specified by systematic names without indication of double bond position, e.g., ‘‘hexadecadienoic acid’’. For Confocal laser scanning electron microscopy quantification, peak areas of fatty acids were determined after baseline correction and normalized to the total peak Confocal microscopy was done with the Zeiss LSM 510 area of all fatty acids. Levels of background contamination with inverted microscope Axiovert 200 at the Life Imaging were determined from chemical blanks, obtained by the Center (LIC, University of Freiburg). The LD LCI Plan- above procedure under omission of biological material, and Apochromat 25x/0.8 DIC ImmKorr water immersion subtracted from sample fatty acid levels. objective was used to search for transformed protoplasts, while the C-Apochromat 63x/1,2 W VIS-IRKorr water Cloning of desaturase-GFP fusion constructs immersions objective was used to take images. For the and protoplast transfection detection of GFP and chlorophyll autofluorescence, the sample was excited with an Argon laser at 488 nm. For For subcellular localization of the fatty acid desaturases, MTO detection a helium-neon laser at 543 nm was moss protoplasts were isolated according to Rother et al. used. Fluorescence signals are false-colored in green (1994) and transiently transfected with desaturase-green (GFP), orange (MTO) and red (chlorophyll), respectively. fluorescent protein (GFP) fusion constructs. The fusion Three-dimensional reconstruction was performed via constructs contained the PpAct5 promoter (Weise et al. z-stacking with the Imaris v3.1 software (Bitplane). 2005) and the coding sequence (CDS) of each fatty acid desaturase, respectively (D5-FADS: Phypa_165175, D6- FADS: Phypa_164045, putative x-3-FADS: Phypa_ Analysis of gene expression 183309), within a GFP-reporter plasmid described before (Kiessling et al. 2004). RNA was extracted from protonema Gene expression analyses of protonema and gametophores with TRIzolÒ reagent (Invitrogen, Karlsruhe, Germany) were performed using a Combimatrix 90 K microarray 123 Plant Cell Rep (2014) 33:245–254 249

Fig. 2 Comparative fatty acid profiles from different mosses. a Fatty acid profiles were established from different moss species (gametophores) that had been cultivated in vitro under axenic conditions. The x-axis depicts the fatty acids written in lipid numbers C:D, where C represents the number of carbon atoms and D the number of double bonds of the fatty acid. The y-axis depicts the relative amount of the fatty acid as a percentage of the total fatty acid content. b Sample GC–MS chromatogram for P. patens, with important fatty acid peaks indicated

(Combimatrix Corp., Mukilteo/WA, USA) based on the (http://www.graphpad.com). Averages and standard devi- v1.2 gene models of P. patens (Rensing et al. 2008)as ations were calculated with Microsoft Excel. described in Wolf et al. (2010). RNA extraction, sample preparation and computational data analysis were done as described previously (Richardt et al. 2010; Wolf et al. Results 2010). The microarray experiments were performed in three biological replicates. Statistical data analyses were Mosses contain high amounts of vlPUFAs done with the Expressionist Analyst 7.5 software (www. genedata.com, Genedata, Basel, Switzerland). The putative The species collection comprised P. patens (Funariaceae), FADS-coding genes were selected based on the KEGG E. streptocarpa (Encalyptaceae), P. lanceolata (Pottia- pathway database (Kanehisa and Goto 2000; Kanehisa ceae), P. undulatum (Mniaceae), B. rutabulum and et al. 2012) using the pathway map ‘‘Biosynthesis of R. murale (), and A. undulatum (Poly- unsaturated fatty acids’’ for P. patens (ppp01040). trichaceae) (Fig. 1). These axenically cultivated moss species contained considerable amounts of vlPUFAs

Statistical analysis ([C18) like AA (20:4), but also smaller amounts of satu- rated very long-chain fatty acids such as tetra- (24:0), To test for significant differences between the fatty acid penta- (25:0) and hexacosanoic acid (26:0) (Fig. 2; Table contents of protonema and gametophores, an unpaired S1). The predominant peak among vlPUFAs was AA in all t-test was performed with the GraphPad software analyzed species (Fig. 2). In P. patens, AA reached a level 123 250 Plant Cell Rep (2014) 33:245–254 of 18.7 % on average in gametophores and 15.9 % in Tissue-specific fatty acid contents correspond protonema (Table 1). Regarding the other species, AA with differential gene expression contents ranged from 6 to 31 % of total fatty acids (Fig. 2). While P. lanceolata and A. undulatum had AA contents of According to our analyses P. patens contains 17.3 % AA in only around 6–10 %, B. rutabulum and R. murale reached gametophores and protonema on average (Table 1). Fur- AA levels of up to 31 % of the total fatty acids (Fig. 2). ther abundant fatty acids ([5 % of total fatty acids) are palmitic acid with an average of 25.9 % in gametophores and protonema, hexadecadienoic acid (16:2) with an average of 5.2 % in both developmental stages, hexadec- Table 1 Highly abundant fatty acids in Physcomitrella patens atrienoic acid (16:3) with 6.2 % in protonema and only 3.3 % in gametophores, LA (18:2) with an average of Fatty acid (C:D, Gametophores (%) Protonema (%) common name) (±SD) (±SD) 12.5 % in gametophores and protonema, linolenic acid (18:3) with an average of 19.4 %, and EPA with 6.8 % in 16:0, Palmitic acid 25.3 (±0.4) 26.5 (±0.04)* protonema (Table 1). The comparative fatty acid profiles 16:2, Hexadecadienoic acid 4.1 (±0.1) 6.2 (±0.5)* revealed significant differences in the abundance of some 16:3, Hexadecatrienoic acid 3.3 (±0.9) 6.2 (±0.3)* fatty acids in the two developmental stages (Table 1; 18:2, Linoleic acid 19.3 (±4.3) 5.8 (±5.3) Fig. 3a). While the saturated fatty acids arachidic acid 18:3, Linolenic acid 14.8 (±1.3) 24.0 (±1.4)* (20:0) and behenic acid (22:0) had a significantly higher 20:4, Arachidonic acid 18.7 (±1.2) 15.9 (±1.4) relative abundance in gametophores, the (poly-)unsaturated 20:5, Eicosapentaenoic acid 1.5 (±0.3) 6.8 (±0.5)* fatty acids hexadecadienoic acid, hexadecatrienoic acid, This table provides an overview of the most abundant fatty acids in P. oleic acid, linolenic acid, dihomo-c-linolenic acid (DGLA) patens. The selection comprises fatty acids with a relative percentage and EPA had a significantly higher abundance in the of all fatty acids that was above 5 % in at least one of the analyzed juvenile protonema stage (Fig. 3a). tissues. Gametophores as well as protonema contain high amounts of Corresponding to the higher relative levels of PUFAs in palmitic acid, followed by linolenic acid in protonema, and linoleic acid in gametophores. Significantly higher levels of a certain fatty protonema than in gametophores, putative D9-, D12-, and acid in one of the two tissues in comparison to the other one are D15-fatty acid desaturase (FADS)-encoding genes also marked with *(p-value \ 0.05, unpaired t-test) showed a higher level of relative gene expression in

Fig. 3 Comparative fatty acid and gene expression profiles from both developmental stages are highlighted in boxes (unpaired t-test, protonema and gametophores of P. patens. a Fatty acid profiles were p-value \ 0.05). b Heat map of the relative gene expression values of established from protonema and gametophores of P. patens. The putative fatty acid desaturase (FADS)-coding genes in P. patens means of fatty acids with abundance higher than 1 % of all fatty acids protonema (P) and gametophores (G) represented in three biological are depicted in the bar chart. The error bars show the standard replicates, respectively deviation. Fatty acids with significantly different abundance between

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Fig. 4 Subcellular localization of the D6- and D5-fatty acid desat- green fluorescence of the GFP tagged to the D6-fatty acid desaturase urase. Physcomitrella patens protoplasts were transformed with a was also visible in the ER, whereas d chlorophyll autofluorescence of plasmid including the CDS of fatty acid desaturase-coding genes and the chloroplasts was localized at distinct positions. e Control staining the coding sequence for green fluorescent protein (GFP). a The green with MitoTrackerÒ Orange CMTMRos (Invitrogen, Karlsruhe, Ger- fluorescence of the GFP tagged to the D5-fatty acid desaturase was many). f Subcellular localization of plastid-localized putative x-3 visible in the endoplasmic reticulum, while b chlorophyll autofluo- fatty acid desaturase. Scale bar 5 lm rescence of the chloroplasts was localized at distinct positions. c The protonema than in gametophores (Fig. 3b). Three of these fluorescence patterns with accumulations and reticular genes (Phypa_22981, Phypa_183309, Phypa_211380) were structures (Fig. 4c), but without co-localization with the significantly higher expressed in protonema than in gameto- fluorescence of chlorophyll (Fig. 4d). We conclude that phores (Benjamini–Hochberg-corrected p-value \ 0.05) both enzymes are localized in the ER. (Benjamini and Hochberg 1995). One putative D12-FADS- The control for localization in mitochondria using Mito- encoding gene (Phypa_22981) was 7.39-fold higher expres- TrackerÒ Orange CMTMRos (MTO, Invitrogen, Karlsruhe, sed in protonema than in gametophores, while two putative Germany) showed mitochondria-specific fluorescence pat- D15-FADS-coding genes were 7.09-fold (Phypa_183309) terns distinct from the fluorescence patterns of the two AA- and 4.70-fold (Phypa_211380) higher expressed in proto- producing FADS:GFP (Fig. 4e). The putative x-3-FADS is nema than in gametophores (Table S2). In accordance to the localized in the chloroplasts, showing co-localization with similar AA contents in gametophores and protonema the fluorescence of the chlorophyll (Fig. 4f), but distinct (Fig. 3a), the AA-producing D5- and D6-FADS-encoding from the fluorescence patterns of the two D6-FADS- and D5- genes showed no significantly deviating gene expression FADS:GFP. levels in the two developmental stages (Fig. 3b).

Arachidonic acid is produced in the endoplasmic Discussion reticulum In this work, we describe seven moss species as rich sources To determine the cellular compartment of AA synthesis, for very long-chain PUFAs. The comparative fatty acid the AA-producing D5- and D6-FADS (Girke et al. 1998; profiles were established from plants grown in axenic in vitro Kaewsuwan et al. 2006) were tagged with green fluorescent culture, a technique that we regard as a prerequisite for protein (GFP). The D5-FADS:GFP showed GFP-fluores- metabolic studies under standardized conditions. All ana- cence in 5 to 10 lm long and 2 lm thick accumulations lyzed mosses contained considerable amounts of arachidonic along with a more reticular weaker fluorescence pattern acid (AA, 20:4D5,8,11,14), a vlPUFA that is usually found in surrounding the nucleus (Fig. 4a). This specific fluores- algae, fish and mammals. According to our analyses, AA is cence pattern was distinct from chlorophyll autofluores- produced in the endoplasmic reticulum (ER) in P. patens. cence (Fig. 4b). The D6-FADS:GFP showed comparable Beside AA smaller amounts of EPA and saturated very 123 252 Plant Cell Rep (2014) 33:245–254

long-chain fatty acids (C22–26) were determined in all ana- question for further research. It is known that PUFAs lyzed mosses. The high content of vlPUFAs in mosses including AA form the precursors of signaling molecules, highlights their potential for biotechnological application. which are collectively named oxylipins (Andreou et al. Especially x-3 PUFAs such as eicosapentaenoic acid 2009; Stumpe et al. 2010; Scholz et al. 2012). Oxylipins (EPA, 20:5D5,8,11,14,17) and docosahexaenoic acid (DHA, are produced by lipid peroxidation based on the enzymatic 22:6D4,7,10,13,16,19) are of importance for human nutrition and activity of lipoxygenases and occur in bacteria, algae, need to be produced in larger amounts, as limited natural plants, fungi and animals (Andreou et al. 2009). In sources basically comprise algae and marine fish (Chodok P. patens oxylipins can be produced from C20 and C18 et al. 2012; Xue et al. 2013). Artificial production of EPA is fatty acids, while in seed plants oxylipins are produced already achieved with metabolic engineering of the yeast from C18 fatty acids only (Wichard et al. 2005; Anterola Yarrowia lipolytica (Xue et al. 2013). However, well-directed et al. 2009). As recently shown for the moss Dicranum modifications of metabolic pathways are also possible in P. scoparium oxylipins possess anti-feeding activity against patens due to its well-annotated genome sequence (Zimmer slugs and contribute to biochemical defense mechanisms et al. 2013) and the high rate of homologous recombination in (Rempt and Pohnert 2010). In P. patens, cyclopentenone– mitotic cells that facilitates the generation of genetically oxylipins, which are precursors of the phytohormone jas- modified strains. This technique enables the production of monic acid in vascular plants, accumulate during pathogen vlPUFAs of interest via metabolic engineering (Kaewsuwan attack by the fungus Botrytis cinerea (Ponce de Leo´n et al. et al. 2010; Chodok et al. 2012). On the other hand, transgenic 2012). Furthermore, cyclopentenone–oxylipins contribute engineering of crops, e.g., oil seed crops using moss genes, as to fertility and sporogenesis of P. patens (Stumpe et al. recently reviewed regarding genes from microalgae or fish 2010). However, the lipid-derived phytohormone jasmonic (Jiao and Zhang 2013) is also a promising research area. acid itself has not been detected in this moss so far The model organism P. patens has already been estab- (Stumpe et al. 2010; Ponce de Leo´n et al. 2012). Con- lished as a production platform for recombinant proteins sidering this, clear differences not only in the lipid and biopharmaceuticals using highly standardized in vitro metabolism, but also in lipid-derived signaling exist cultivation techniques in photobioreactors (Decker and between mosses and higher plants. The high contents of Reski 2012). However, the opportunity of metabolic vlPUFAs may represent a key physiological characteristic engineering along with cultivation under highly standard- that contributes to the considerable biotic and abiotic ized conditions represents one step further towards the stress tolerance of mosses. biotechnological use of mosses as PUFA sources under good manufacturing practice (GMP) conditions. Recently, Acknowledgments This work was supported by the Deutsche the C -PUFAs adrenic acid (ADA, 22:4D7,10,13,16) and Forschungsgemeinschaft (DFG, GRK 1305), the MOSSCLONE FP7- 22 ENV.2011.3.1.9-1 and the Excellence Initiative of the German Fed- the DHA-precursor x-3 docosapentaenoic acid (DPA, eral and State Governments (EXC294 to Ralf Reski). We are grateful 22:5 D7,10,13,16,19) were produced in P. patens by heterol- to Jan-Peter Frahm for his help with the classification of the moss ogous expression of a D5-elongase from a marine alga species, the team of the Life Imaging Center Freiburg and Dr. Stef- (Kaewsuwan et al. 2010; Chodok et al. 2012). Considering anie Mu¨ller for their help with confocal microscopy and Anne Katrin Prowse for proofreading of the manuscript. the biotechnological techniques available, an increased production of the x-3 fatty acids EPA or DHA might also Conflict of interest The authors declare that they have no conflict be possible in P. patens and other mosses. of interest. However, it should be taken into account that fatty acid profiles from different developmental stages showed Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, dis- remarkable differences with regard to PUFA contents in tribution, and reproduction in any medium, provided the original P. patens. These findings are in accordance with the pre- author(s) and the source are credited. viously reported distinct metabolic profiles of protonema and gametophores regarding saccharides, sugar derivates, amino acids and nitrogen-rich storage compounds (Erxle- ben et al. 2012). According to our analyses, the relative References amounts of PUFAs were higher in protonema than in ga- metophores, a finding that is supported by the significantly Andreou A, Brodhun F, Feussner I (2009) Biosynthesis of oxylipins increased expression of putative fatty acid desaturase in non-mammals. Prog Lipid Res 48:148–170 (FADS)-encoding genes in protonema when compared Anterola A, Go¨bel C, Hornung E, Sellhorn G, Feussner I, Grimes H (2009) Physcomitrella patens has lipoxygenases for both eicos- with the gene expression level in gametophores. anoid and octadecanoid pathways. Phytochemistry 70:40–52 The biological meaning of the higher PUFA levels in Asakawa Y (2007) Biologically active compounds from bryophytes. protonema in comparison to gametophores remains a Pure Appl Chem 79:557–580 123 Plant Cell Rep (2014) 33:245–254 253

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